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Lipoxidase and the oxygen absorption of homogenates from corn seedlings
Lipoxidase and the Oxygen Absorption of Homogenates from Corn Seedlings’ George Fritz and Harry Beevers Prom the Department
of Biological Sciences, Lafayette, Zndiana
Purdue
University,
Received July 23, 1954 INTRODUCTION
The enzyme lipoxidase catalyzes the peroxidation of linoleic, linolenic, and arachidonic acids and their esters [for a review see Holman and Bergstrijm (7)]. This nonmetallic enzyme has been crystallized from soybean and de&ted in the tissues of other higher plants, but has not been reported from corn. During the (aourse of an investigation of the t.erminal oxidases of corn seedlings, it was found t,hat, homogenates prepared from 2- to 4day et.iolated seedlings absorbed oxygen rapidly. Further investigation revealed that the enzyme lipoxidase and it,s substrates were present and that this enzyme system accounted for a large portion of the oxygen absorpt,ion of the homogenate. MATERIAL
AKD METHODS
Manometric experiments were performed with stnndard Warburg equipment at 25’C.; the total volume of reactants in each flask was 2.5 ml., and KOH-saturated paper was placed in the center well except in certain inhibitor experiments as noted. (When homogenates were used, some CO? output occurred, since omission of KOH papers resulted in an apparent decrease approximating 20% in the rate of 02 absorption; in experiments with the derived fractions, there was no apparent CO* output.) For spectrophotometric determinations, a Beckman model DU instrument was used, with silica cuvettes of l-ml. light path. Centrifugation was carried out at room temperature and the centrifugal force applied was 15,000 g. Emulsions of the fatty acids and of the inhibitora-naphthol were prepared with gum ghatti solution in the manner described by Holman (6). 1 From the dissertation of George Fritz submitted to the Graduate School of Purdue University in partial fulfillment. of the requirements for the Ph.D. degree. This research was supported by a grant from tha Purdue Research Foundation. 436
I,IPOX.IDhSE
AND
OXYGEN
-4BSORPTION
437
Plant Material Grain of corn hybrid Wf9 x 38-11 was soaked in tap water for 6 hr. and then distributed upon mosquito netting supported between two steel screens, which were placed over a glass tray containing tap water; a wet clot.h, the edges of which dipped into the water of the outside tray, was spread over the topmost screen. The soaking and subsequent germination and growth took place at 29°C. in a dark room with occasional red light. The tap water bathing the roots was not changed (except when plants were grown for more than 3 days, in which case the water was changed daily after the third day). Upon harvesting, the endosperm was removed, so that, the experimental material consisted of the shoot-root axis and attached scutellum. The tissue was washed in tap water and dried with papet toweling. If the material was not used immediately, it was stored at 10°C. for one to several hours before use. (For certain experiments, as noted, the tissue was frozen overnight and thawed before use.)
Preparation of Homogenate The weighed mat.erial was ground with mortar and pestle with half its weight of 0.1 M phosphate buffer (pH 7.7) ; a small quantity of white sand was sprinkled on the tissue to facilitate grinding. The mash was strained through a piece of broadcloth and the recovered plant juice constituted the homogenate. The pH was adjusted, if required, with concentrated HCl or NaOH. The plant, juice was used as soon as it was extracted, whether for manometric or spectrophotomet,ric experiments or for further treatment, such as centrifugation. RESULTS
AND
DISCUSSION
Endogenous Oxygen Absorption of Corn Homogenates Homogenates prepared as outlined above absorbed oxygen actively, and boiling of the extract reduced the absorption to 5 yO of the original \-aiue. The rat.e of oxygen uptake was found to depend upon (a) t,he age of the seedlings from which the homogenates were prepared and (b) the pH of the homogenate. The first effect is shown in Fig. 4, curve 1, which records the rate of oxygen uptake of homogenates of corn seedlings of various ages, measured within one-half hour aft,er preparation. (The pH of the plant extract varied from 6.5 t,o 6.8, increasing with the age of t.he seedlings; no adjust(ment of pH was made in this experiment.) Homogenates prepared from seedlings about 3 days old absorbed oxygen much more rapidly t’han homogenates derived from seedlings of other ages. For practical reasons involving the time of day at which seedlings were first placed into water, t,he work reported in this paper \vas done with 2f,i-day seedlings. The effect of pH of the homogenate upon t,he rate of oxygen absorption of homogenates from 2l,i-day corn seedlings is shown in Fig. 1,
438
GEORGE
FRITZ
.4ND
Ht\RRY
BEEVERS
160-
B g 40b I+ d
(3) 45
5.5
6.5
I 7.5
pH OF HOMOGENATE
Fro. 1. The effect of pH of the homogenate prepared from ax-day corn scedlings upon its rate of oxygen absorption. When first extracted from the seedlings, the pH waa 6.5 and was adjusted to other pII values with concentrated acid or alkali. (Flask contents: 1.0 ml. homogenate, 1.5 ml. water, KOII-saturated paper in center well.) Curve 1: Homogenate prepared from freshly harvested seedlings. Curve 2: Homogenate prepared from seedlings frozen overnight. Curve 3: Homogenate prepared from seedlings which were lyophilized and ether extracted before homogenization (see text for details).
curve 1. The pH of the homogenate was 6.5 upon extraction, and was adjusted to various pH levels with concentrated HCl or KaOH. Figure 1 shows that two maxima were obtained for homogenates from freshly harvested seedlings, one at pH 5.0 and the other at pH 7.2 (curve 1). When the tissue was frozen overnight and thawed before grinding, only one maximum was obtained, at pH 5.8 (Fig. 1, curve 2). It would appear that freezing the tissue resulted in a shifting of the lower maximum to a higher pH value, and a disappearance of the upper maximum. Detection of Lipoxidase and Substrates in the Homogenate When the homogenate from 2x-day corn seedlings (frozen overnight and thawed) was adjusted to pH 4.8 with concentrated HCl and centri-
LIPOXIDASE
AND
OXYGEN
ABSORPTION
439
fuged at room temperature for 15-20 min. at a force of 15,000 g., the homogenate separated into two distinct fractions. The supernatant liquid was straw-colored and constituted about three-quarters of the volume of the homogenate. The oxygen absorption of these supernatant fractions was barely detectable manometrically, either before or after adjustment of the pH to 7.0. During this pH adjustment, a white precipitate formed, which could be separated from the supernatant fraction by centrifugation. When the supernatant was stored, a browning slowly occurred due to the presence of an autoxidizable pigment. The sediment fraction alone absorbed small amount,s of oxygen; by resuspending in 0.1 M phosphate buffer and re-centrifuging at pH 4.8, the oxygen absorption of the recovered sediment was reduced, and after several washings it was virtually zero. However, the addition of the washed sediment to the supernatant resulted in a rapid oxygen absorption, with a pH optimum at 7.5. Autoclaving of each fraction separately showed t’hat t,he supernatant fraction was thermolabile and contained an enzyme which catalyzed oxygen absorption upon addition of the sediment fraction, which itself was not inactivated by t’he heat treatment. (The white precipitate formed during the neutralization of the supernatant, referred to above, was inactive and was discarded.) The abilit,y of the supernatant to induce oxygen upt’ake in the presence of the sediment was lost’ if the sediment was ext,racted with acidified ether. However, when the gummy residue remaining after evaporation of the ether was emulsified in water, a rapid oxygen absorption occurred at pH 7.0 when it was added to the supernatant (enzyme) fraction. Inasmuch as the natural substrate in the sediment fraction was very soluble in ether, this suggested the possibility that the unknown enzyme in t,he supernatant was lipoxidase, and it was in fart found that when an rmulsion of linoleic arid was added t#o the supernatant enzyme, a rapid oxygen ahsorpt’ion occurred. The proof that lipoxidase was present in the supernatant was based upon the work of Theorell, Hergst’riim, and Wkeson (1 I), who showed that the products of fatt,y acid oxidation absorb strongly at 234 rnp, and upon t’he work of Holman ((i), who showed that the development of an absorption maximum at 234 rnp in a fatty acid undergoing oxidation is paralleled by oxygen absorption. Figure 2 (curve 1) shows the course of oxygen absorption of methyl linoleate in the presence of t’he supernatant, from a homogenate from 21,i-day corn seedlings. Curve 2 shows the optical densities at, 234 rnF of aliquots of the reaction mixture used to obtain the data of curve 1; t,he met.hod used
440
OEORQE
FRITZ
AND
HARRY
BEEVERS
to prepare solutions of methyl linoleate suit,ahle for spectrophotometric measurements involves dissolving the fatty acid in methanol, and is that recommended by Holman (6). A similar procedure was used to prove that lipoxidase substrates mere present in the sediment fraction. When the sediment. was suspended in phosphate buffer (pH 7.0) and autoclaved, no oxygen was absorbed until lipoxidase (from the supernatant fraction) was added. Furthermore, when t,he supernatant was added to the sediment, t.he resulting oxygen absorption was paralleled by an increase in optical density at 234 rnp of methanol extracts of aliyuots of t.he digest.. Finally, t,he addition of lipoxidase ohtained from soyhean (Worthington Biochemical Corporat.ion, Freehold, K. .J.) induced oxygen absorption by the aut.oclavcd sediment fraction. Consequently it could be concluded that lipoxidase and/or arachidonic acids and their substrates-linolek, linolenir, esters--were present in t.he sediment.
0
5
I5
0 TIME
23
2
(MIN.)
Ftc.. 2. Curve 1. The oxygen absorption of a methyl linolentr emulsion catalyzed by the supernatant frnct.ion from rorn-seedling homogenlite. Cont.enta of flask: 1.5 ml. methyl linoleate emulsion, 0.5 ml. supernutant eolution, 0.5 ml. 0.1 M phosphate buffer (pII 7.0). Curve 2. Optical densities at 231 rnr of methanol extracts of aliquots of the reaction mixture taken at the times indicated.
LIPOXIDASE
AND
OXYGEN
ABSORPTION
441
The Relation of the Lipoxidase-Substrate System to the Totul Oxygen Absorption of the Homogenate When it was evident that lipoxidase and its substrates mere present in homogenates from 21/4-day corn seedlings, it became of interest to determine the extent to which this system participated in oxygen absorption. Figure 3 shows the results of experiments in which two homogenate samples-one at pH 5.0 and one at pH 7.2, corresponding to the two peaks in curve 1, Fig. l- were extracted with methanol (6) and the methanol solutions examined at 234 mp. For the homogenate at pH 5.0
160-
TIME
(MIN.)
FIG. 3. Curve 1A. The oxygen absorption of the homogenate from ax-day freshly harvest,ed corn seedlings at pH 5.0. (Flask contents: 1.0 ml. homogenate, 1.5 ml. water, KOH-saturated paper in center well.) Curve 1B. Optical densities at 234 rns of methanol extracts of aliquots of the homogenate of curve IA, taken at the times indicated. Curve 2A. The oxygen absorption of the homogenate from ax-day freshly harvested corn seedlings at pH 7.2. (Flask contents same as in curve 1A.) Curve 2B. Optical densities at 234 w of methanol extract.8 of aliquots of the homogenate of curve 2A, taken at the times indicated.
442
GEORGE
FRITZ
AND HARRY
BEEVERS
(Fig. 3, curves 1A and lB), there was a very pronounced increase in optical density accompanying oxygen absorption, which would be expected if the homogenate was oxidizing lipoxidase substrates (6, 11). But at pH 7.2 (Fig. 3, curves 2A and 2B), where the oxygen uptake was much lower during the same time interval, t.he increase in optical density at 234 rnp was small, indicating that lipoxidase substrates in the homogenate probably were not oxidized vigorously at this pH value. Experiments with inhibitors confirmed and extended these findings. Application of compounds known to inhibit lipoxidasc (7)---y-naphthol, catechol, and hydroquinone-at concentrations of 0.01 M almost completely inhibited oxygen absorption of the homogenate at pH 5.0, but not at pH 7.2 (Table I), indicating that the enzymatic oxygen absorption of the homogenate at pH 5.0 was due almost entirely to the lipoxidasesubstrate system. On the other hand, application of the metallic inhibitors cyanide, azide, and dicthyl dithiocarbamate at pH 5.0 showed t.hat only diethyl dithiocarbamate inhibited oxygen absorption to a small extent; at pH 7.2, only cyanide was effective (Table I). (KOH papers were omitted from the center well for the inhibition experiments with cyanide and azide.) Further evidence that the lipoxidase-substrate system was responsible for a large portion of the oxygen uptake of the homogenate, particularly at pH 5.0, w&s obtained by removing lipoxidase substrates from corn seedlings by ether extraction. For this purpose, freshly harvested 214day corn seedlings were dehydrated by lyophilizing and exhaustively extracted with ether. The resulting powder was wetted with the amount TABLE Inhibiton
of Oxygen
I
Absorption of 2x-Day Corn-Seedling and at pH 7.2 by Various Inhibitors
(The concentration
of the inhibitors
Homogenate
at pH 6.0
was 0.01 M) Per cent inhibition
Inhibitor At pH 5.0
At pH 7.2
0 0 25 95 98 95
0 50 0 0 50 25
__
Sodium azide Sodium cyanide Diethyl dithiocarbamate a-Naphthol Catechol Hydroquinone _..-
LIPOXIDASE
AND
OXYGEN
ABSORPTION
443
of water lost during drying. This powder and water mixture was treated as if it was fresh material, and a homogenate was prepared in the manner already described. In contrast to fresh homogenates (Fig. 1, curve l), such homogenates had low rates of endogenous oxygen uptake at all pH levels (Fig. 1, curve 3); but when linoleic acid emulsion was added at pH 7.0, a rate of oxygen uptake occurred which was even higher than that produced when linoleic acid was added to the fresh homogenate. It could thus be concluded that the ether extraction and freezing treatments had no adverse effect upon the lipoxidase, but effectively removed the native substrates. When the results of this experiment are considered in conjunction with the demonstration of increased optical absorption at 234 rnp during oxygen absorption of the homogenate at pH 5.0 (Fig. 3, curves 1A and lB), and the results of the inhibition experiments (Table I), they strongly support the view that t’he oxygen absorption of the homogenate at pH 5.0 is due almost entirely t.o the lipoxidasesubstrate system. For the homogenate at pH 7.2, the inhibition of oxygen absorption by cyanide (Table I) indicated the participation of certain metallic oxidases in the oxygen uptake at this pH level. Cytochrome oxidase was detected in the homogenates from 214-day corn seedlings manometrically, using p-phenylenediamine as reductant (IO), and spectrophotometrically (2). However, since the homogenate itself was insensitive to azide (Table I), it is probable that cytochrome oxidase, even though present, does not contribute to the endogenous oxygen absorption of the homogenate. Phenoloxidase was also detected in homogenates from corn seedlings, but not at all ages. Using the thermolabile oxygen uptake induced by catechol as a measure of phenoloxidase, no activity was detected in homogenates from l-, 2-, and S-day corn seedlings. However, oxidation of catechol was rapidly catalyzed by homogenates from older seedlings (Fig. 4). Also, the production of intensely red compounds was observed during the course of oxygen absorption, a phenomenon associated with non-enzymatic reactions of quinoid products resulting from the oxidation of catechol (1, 3). This reddish pigment was faintly discernible with the use of homogenates from 2- or 3-day corn seedlings. Since phenoloxidase appears to be present only in small amounts in homogenates from such seedlings, and since oxygen uptake of homogenates was not strongly inhibited by diethyl dithiocarbamate (Table I), an inhibitor of this enzyme (8), it is unlikely that phenoloxidase is a major terminal oxidase in endogenous oxygen uptake of homogenates of 2x-day corn
444
GEORGE
FRITZ
AND
HARRY
BEEVERS
1507 .E 9 > 3
-
y IOO!T fj 0 508 P UQ
AGE OF SEEDLINGS (DAYS) Fm 4. Curve 1. The rate of oxygen absorption of homogenates from corn seedlings l-6 days old. The pH of the homogenates varied from 6.5 to 6.8, increasing with the age of the seedling. (One milliliter of the homogenate was used per flask, and KOH-saturated paper was in the center well.) Curve 2. The rate of oxygen absorption of the homogenates of curve 1 in the presence of 0.2 M catechol.
seedlings; however, the possibility is not excluded that phenoloxidase plays a greater role at later stages of development,. Still other metallic oxidases may contribute to the oxygen absorption of the homogenate at, pH 7.2. Although homogenates from corn seedlings can oxidize ascorbic acid, no conclusion was reached regarding the possible presence of ascorbic oxidase, since ascorbic acid can be oxidized through more than one enzyme system, such as cytochrome oxidase and phenoloxidase, as well as ascorbic oxidase (5, 9). The work of Ginter and Smith (4) indicated that a new type of oxidative enzyme may be present in homogenates from the tips of corn roots. It should be stressed that the conclusion that lipoxidase plays a major role in oxygen absorption at pH 5.0 applies only to the homogenates; it is not suggested that lipoxidase participates in the same degree in oxygen uptake of the intact tissue. Indeed, it was found that the respiration of 2x-day corn tissue was inhibited to the extent, of about, 50 % by application of 0.01 M cyanide. The clarification of the role of lipoxidase in the remaining (cyanide-insensitive) respiration must await further study.
LIPOXIDASE
The pH Optimum
AND
OXYGEN
-4BSORPTION
of the Lipoxidase-Catalyzed
Reaction
Holman and Bergstriim (7) have discussed the difficulties of determining the pH optimum of lipoxidase. Since the substrates for lipoxidase are difficultly soluble in water and not equally available to t,he enzyme at’ all pH \Talues, they concluded that the effect of pH upon (a) the inherent, enzyme activity and (b) the availability of t,he substrate to the enzyme cannot be separat,ed so long as emulsified substrates, not equally soluble over the entire pH range in question, are used. It, has already been shown that the lipoxidasa system a,rcounts for the bulk of the endogenous oxidative activity of homogenates from 21,i-day corn seedlings, and t)he pH optimum occurred at pH 5.0 (Fig. I, curve 1). However, when the homogenate was centrifuged (as previously described) and separated into two fractions, one of which contained lipoxidase and the other the substrates of lipoxidase, the pH optimum for the oxygen absorption process of the mixture of the two fractions occurred at pH 7.5, and very little oxygen was absorbed at pH 5.0. (When linoleic acid emulsion was added either to the supernatant or to the original (diluted) homogenate, the pH optimum was again 7.5.) Even if t)he sediment fraction was autoclaved, or subjected to wide cahanges in acidity before addition to the supernatant fract’ion containing lipoxidase, the pH optimum was still at pH 7.5 after mixture of the two fractions. Consequently, it was concluded that the centrifugation process itself was responsible for the loss of activity at pH 5.0, and the shift’ in optimum activity to pH 7.5. (In this connection, it may be noted that freezing and t,hawing of the corn seedlings had a similar effect, in t,hat t,hc optimum act,ivity had been shifted to a higher pH value; see Fig. 1, curve 2.) It is suggested that an intimate union between lipoxidase and its sub&rates exists in the homogenate prepared from fresh tissue, and accounts for the rapid endogenous oxygen absorption of the homogenate at pH 5.0. It would appear that this union is destroyed by physical treatments such as centrifugation, and is not completely restored upon subsequent, mixing of the enzyme and iDs substrat’es. (Probably the freezing and thawing process also partially disrupts t,his union.) Therefore it is suggested that the optimum pH value of the lipoxidase-catalyzed reaction in corn-seedling homogenates is pH 5.0. The fact that the pH optimum was found to be 7.5 when exogenous suhstrat,c was added, whether to t,he (diluted) homogenat,e or to the supernatant fraction, would be a result of t,he greater availability (nolubility) of the added substrate at t,he higher pH, but would not be a measure of the inherent
446
GEORGE
FRITZ
AND HhRRY
BEEVERS
pH optimum of the enzyme, a viewpoint already discussed by Holman and Bergstrijm (7). These comments indicate the need for distinguishing between native substrate already present and in closest possible contact with the enzyme, and exogenous or added substrate, limited in availability to the enzyme by its solubility. SUMMARY
Homogenates from 2x-day corn seedlings absorbed oxygen most rapidly at pH 5.0 and a second smaller maximum occurred at pH 7.2. The homogenat.es could be separated into t.wo fractions, one of which contained lipoxidase and the other contained substrates for lipoxidase. Several lines of evidence led to the conclusion that the lipoxidase-substrate system accounts for the major portion of the oxygen absorption of the homogenate at pH 5.0 but at pH 7.2, other enzymes systems must contribute to the oxygen absorption. REFERENCES 1. BEEVERS, H., ASD JAMES, W. O., Biochern. J. 43,636 (1948). 2. COOPERSTEIS, S. J., AND LAZAROW, A., J. Biol. Chem. 189, 665 (1951). 3. LAWSON, C. R., ASD TARPLEY, W. B., in “The Enzymes” (.I. B. Sumnerand K. Myrblick, eds.), Vol. II. .4cademic Press, New York, 1951. 4. GISTER, E. L., ASD SMITH, F. G., Zuwa Slate (‘011. J. Sci. 28, 177 (1953). 5. HII.L, R., AND HARTREE, E. F., Ann. Rev. Plant Physiol. 4, 115 (1953). 6. IIOLMAS, R. T., Arch. Biochem. 10, 519 (1946). 7. HOLMAN, R. T., ASD BERGSTRBM, S., in “The Enzymes” (J. B. Sumner and K. Myrb&ck, eds.), Vol. II. Academic Press, New York, 1951. 8. JAMES, W. O., Ann. Rev. Planl Physiol. 4, 59 (1953). 9. M~rsoN, I,. W., I’itamins and Hormones 11.1 (1953). 10. SLATEH, E. c., Biochem. .I. 44, 305 (1949). 11. THEORELL, H., RERGSTR~~M, S., .~SD AKESON, .4., Pharm. Acln Helv. 21, 318 (1946).
of Biological Sciences, Lafayette, Zndiana
Purdue
University,
Received July 23, 1954 INTRODUCTION
The enzyme lipoxidase catalyzes the peroxidation of linoleic, linolenic, and arachidonic acids and their esters [for a review see Holman and Bergstrijm (7)]. This nonmetallic enzyme has been crystallized from soybean and de&ted in the tissues of other higher plants, but has not been reported from corn. During the (aourse of an investigation of the t.erminal oxidases of corn seedlings, it was found t,hat, homogenates prepared from 2- to 4day et.iolated seedlings absorbed oxygen rapidly. Further investigation revealed that the enzyme lipoxidase and it,s substrates were present and that this enzyme system accounted for a large portion of the oxygen absorpt,ion of the homogenate. MATERIAL
AKD METHODS
Manometric experiments were performed with stnndard Warburg equipment at 25’C.; the total volume of reactants in each flask was 2.5 ml., and KOH-saturated paper was placed in the center well except in certain inhibitor experiments as noted. (When homogenates were used, some CO? output occurred, since omission of KOH papers resulted in an apparent decrease approximating 20% in the rate of 02 absorption; in experiments with the derived fractions, there was no apparent CO* output.) For spectrophotometric determinations, a Beckman model DU instrument was used, with silica cuvettes of l-ml. light path. Centrifugation was carried out at room temperature and the centrifugal force applied was 15,000 g. Emulsions of the fatty acids and of the inhibitora-naphthol were prepared with gum ghatti solution in the manner described by Holman (6). 1 From the dissertation of George Fritz submitted to the Graduate School of Purdue University in partial fulfillment. of the requirements for the Ph.D. degree. This research was supported by a grant from tha Purdue Research Foundation. 436
I,IPOX.IDhSE
AND
OXYGEN
-4BSORPTION
437
Plant Material Grain of corn hybrid Wf9 x 38-11 was soaked in tap water for 6 hr. and then distributed upon mosquito netting supported between two steel screens, which were placed over a glass tray containing tap water; a wet clot.h, the edges of which dipped into the water of the outside tray, was spread over the topmost screen. The soaking and subsequent germination and growth took place at 29°C. in a dark room with occasional red light. The tap water bathing the roots was not changed (except when plants were grown for more than 3 days, in which case the water was changed daily after the third day). Upon harvesting, the endosperm was removed, so that, the experimental material consisted of the shoot-root axis and attached scutellum. The tissue was washed in tap water and dried with papet toweling. If the material was not used immediately, it was stored at 10°C. for one to several hours before use. (For certain experiments, as noted, the tissue was frozen overnight and thawed before use.)
Preparation of Homogenate The weighed mat.erial was ground with mortar and pestle with half its weight of 0.1 M phosphate buffer (pH 7.7) ; a small quantity of white sand was sprinkled on the tissue to facilitate grinding. The mash was strained through a piece of broadcloth and the recovered plant juice constituted the homogenate. The pH was adjusted, if required, with concentrated HCl or NaOH. The plant, juice was used as soon as it was extracted, whether for manometric or spectrophotomet,ric experiments or for further treatment, such as centrifugation. RESULTS
AND
DISCUSSION
Endogenous Oxygen Absorption of Corn Homogenates Homogenates prepared as outlined above absorbed oxygen actively, and boiling of the extract reduced the absorption to 5 yO of the original \-aiue. The rat.e of oxygen uptake was found to depend upon (a) t,he age of the seedlings from which the homogenates were prepared and (b) the pH of the homogenate. The first effect is shown in Fig. 4, curve 1, which records the rate of oxygen uptake of homogenates of corn seedlings of various ages, measured within one-half hour aft,er preparation. (The pH of the plant extract varied from 6.5 t,o 6.8, increasing with the age of t.he seedlings; no adjust(ment of pH was made in this experiment.) Homogenates prepared from seedlings about 3 days old absorbed oxygen much more rapidly t’han homogenates derived from seedlings of other ages. For practical reasons involving the time of day at which seedlings were first placed into water, t,he work reported in this paper \vas done with 2f,i-day seedlings. The effect of pH of the homogenate upon t,he rate of oxygen absorption of homogenates from 2l,i-day corn seedlings is shown in Fig. 1,
438
GEORGE
FRITZ
.4ND
Ht\RRY
BEEVERS
160-
B g 40b I+ d
(3) 45
5.5
6.5
I 7.5
pH OF HOMOGENATE
Fro. 1. The effect of pH of the homogenate prepared from ax-day corn scedlings upon its rate of oxygen absorption. When first extracted from the seedlings, the pH waa 6.5 and was adjusted to other pII values with concentrated acid or alkali. (Flask contents: 1.0 ml. homogenate, 1.5 ml. water, KOII-saturated paper in center well.) Curve 1: Homogenate prepared from freshly harvested seedlings. Curve 2: Homogenate prepared from seedlings frozen overnight. Curve 3: Homogenate prepared from seedlings which were lyophilized and ether extracted before homogenization (see text for details).
curve 1. The pH of the homogenate was 6.5 upon extraction, and was adjusted to various pH levels with concentrated HCl or KaOH. Figure 1 shows that two maxima were obtained for homogenates from freshly harvested seedlings, one at pH 5.0 and the other at pH 7.2 (curve 1). When the tissue was frozen overnight and thawed before grinding, only one maximum was obtained, at pH 5.8 (Fig. 1, curve 2). It would appear that freezing the tissue resulted in a shifting of the lower maximum to a higher pH value, and a disappearance of the upper maximum. Detection of Lipoxidase and Substrates in the Homogenate When the homogenate from 2x-day corn seedlings (frozen overnight and thawed) was adjusted to pH 4.8 with concentrated HCl and centri-
LIPOXIDASE
AND
OXYGEN
ABSORPTION
439
fuged at room temperature for 15-20 min. at a force of 15,000 g., the homogenate separated into two distinct fractions. The supernatant liquid was straw-colored and constituted about three-quarters of the volume of the homogenate. The oxygen absorption of these supernatant fractions was barely detectable manometrically, either before or after adjustment of the pH to 7.0. During this pH adjustment, a white precipitate formed, which could be separated from the supernatant fraction by centrifugation. When the supernatant was stored, a browning slowly occurred due to the presence of an autoxidizable pigment. The sediment fraction alone absorbed small amount,s of oxygen; by resuspending in 0.1 M phosphate buffer and re-centrifuging at pH 4.8, the oxygen absorption of the recovered sediment was reduced, and after several washings it was virtually zero. However, the addition of the washed sediment to the supernatant resulted in a rapid oxygen absorption, with a pH optimum at 7.5. Autoclaving of each fraction separately showed t’hat t,he supernatant fraction was thermolabile and contained an enzyme which catalyzed oxygen absorption upon addition of the sediment fraction, which itself was not inactivated by t’he heat treatment. (The white precipitate formed during the neutralization of the supernatant, referred to above, was inactive and was discarded.) The abilit,y of the supernatant to induce oxygen upt’ake in the presence of the sediment was lost’ if the sediment was ext,racted with acidified ether. However, when the gummy residue remaining after evaporation of the ether was emulsified in water, a rapid oxygen absorption occurred at pH 7.0 when it was added to the supernatant (enzyme) fraction. Inasmuch as the natural substrate in the sediment fraction was very soluble in ether, this suggested the possibility that the unknown enzyme in t,he supernatant was lipoxidase, and it was in fart found that when an rmulsion of linoleic arid was added t#o the supernatant enzyme, a rapid oxygen ahsorpt’ion occurred. The proof that lipoxidase was present in the supernatant was based upon the work of Theorell, Hergst’riim, and Wkeson (1 I), who showed that the products of fatt,y acid oxidation absorb strongly at 234 rnp, and upon t’he work of Holman ((i), who showed that the development of an absorption maximum at 234 rnp in a fatty acid undergoing oxidation is paralleled by oxygen absorption. Figure 2 (curve 1) shows the course of oxygen absorption of methyl linoleate in the presence of t’he supernatant, from a homogenate from 21,i-day corn seedlings. Curve 2 shows the optical densities at, 234 rnF of aliquots of the reaction mixture used to obtain the data of curve 1; t,he met.hod used
440
OEORQE
FRITZ
AND
HARRY
BEEVERS
to prepare solutions of methyl linoleate suit,ahle for spectrophotometric measurements involves dissolving the fatty acid in methanol, and is that recommended by Holman (6). A similar procedure was used to prove that lipoxidase substrates mere present in the sediment fraction. When the sediment. was suspended in phosphate buffer (pH 7.0) and autoclaved, no oxygen was absorbed until lipoxidase (from the supernatant fraction) was added. Furthermore, when t,he supernatant was added to the sediment, t.he resulting oxygen absorption was paralleled by an increase in optical density at 234 rnp of methanol extracts of aliyuots of t.he digest.. Finally, t,he addition of lipoxidase ohtained from soyhean (Worthington Biochemical Corporat.ion, Freehold, K. .J.) induced oxygen absorption by the aut.oclavcd sediment fraction. Consequently it could be concluded that lipoxidase and/or arachidonic acids and their substrates-linolek, linolenir, esters--were present in t.he sediment.
0
5
I5
0 TIME
23
2
(MIN.)
Ftc.. 2. Curve 1. The oxygen absorption of a methyl linolentr emulsion catalyzed by the supernatant frnct.ion from rorn-seedling homogenlite. Cont.enta of flask: 1.5 ml. methyl linoleate emulsion, 0.5 ml. supernutant eolution, 0.5 ml. 0.1 M phosphate buffer (pII 7.0). Curve 2. Optical densities at 231 rnr of methanol extracts of aliquots of the reaction mixture taken at the times indicated.
LIPOXIDASE
AND
OXYGEN
ABSORPTION
441
The Relation of the Lipoxidase-Substrate System to the Totul Oxygen Absorption of the Homogenate When it was evident that lipoxidase and its substrates mere present in homogenates from 21/4-day corn seedlings, it became of interest to determine the extent to which this system participated in oxygen absorption. Figure 3 shows the results of experiments in which two homogenate samples-one at pH 5.0 and one at pH 7.2, corresponding to the two peaks in curve 1, Fig. l- were extracted with methanol (6) and the methanol solutions examined at 234 mp. For the homogenate at pH 5.0
160-
TIME
(MIN.)
FIG. 3. Curve 1A. The oxygen absorption of the homogenate from ax-day freshly harvest,ed corn seedlings at pH 5.0. (Flask contents: 1.0 ml. homogenate, 1.5 ml. water, KOH-saturated paper in center well.) Curve 1B. Optical densities at 234 rns of methanol extracts of aliquots of the homogenate of curve IA, taken at the times indicated. Curve 2A. The oxygen absorption of the homogenate from ax-day freshly harvested corn seedlings at pH 7.2. (Flask contents same as in curve 1A.) Curve 2B. Optical densities at 234 w of methanol extract.8 of aliquots of the homogenate of curve 2A, taken at the times indicated.
442
GEORGE
FRITZ
AND HARRY
BEEVERS
(Fig. 3, curves 1A and lB), there was a very pronounced increase in optical density accompanying oxygen absorption, which would be expected if the homogenate was oxidizing lipoxidase substrates (6, 11). But at pH 7.2 (Fig. 3, curves 2A and 2B), where the oxygen uptake was much lower during the same time interval, t.he increase in optical density at 234 rnp was small, indicating that lipoxidase substrates in the homogenate probably were not oxidized vigorously at this pH value. Experiments with inhibitors confirmed and extended these findings. Application of compounds known to inhibit lipoxidasc (7)---y-naphthol, catechol, and hydroquinone-at concentrations of 0.01 M almost completely inhibited oxygen absorption of the homogenate at pH 5.0, but not at pH 7.2 (Table I), indicating that the enzymatic oxygen absorption of the homogenate at pH 5.0 was due almost entirely to the lipoxidasesubstrate system. On the other hand, application of the metallic inhibitors cyanide, azide, and dicthyl dithiocarbamate at pH 5.0 showed t.hat only diethyl dithiocarbamate inhibited oxygen absorption to a small extent; at pH 7.2, only cyanide was effective (Table I). (KOH papers were omitted from the center well for the inhibition experiments with cyanide and azide.) Further evidence that the lipoxidase-substrate system was responsible for a large portion of the oxygen uptake of the homogenate, particularly at pH 5.0, w&s obtained by removing lipoxidase substrates from corn seedlings by ether extraction. For this purpose, freshly harvested 214day corn seedlings were dehydrated by lyophilizing and exhaustively extracted with ether. The resulting powder was wetted with the amount TABLE Inhibiton
of Oxygen
I
Absorption of 2x-Day Corn-Seedling and at pH 7.2 by Various Inhibitors
(The concentration
of the inhibitors
Homogenate
at pH 6.0
was 0.01 M) Per cent inhibition
Inhibitor At pH 5.0
At pH 7.2
0 0 25 95 98 95
0 50 0 0 50 25
__
Sodium azide Sodium cyanide Diethyl dithiocarbamate a-Naphthol Catechol Hydroquinone _..-
LIPOXIDASE
AND
OXYGEN
ABSORPTION
443
of water lost during drying. This powder and water mixture was treated as if it was fresh material, and a homogenate was prepared in the manner already described. In contrast to fresh homogenates (Fig. 1, curve l), such homogenates had low rates of endogenous oxygen uptake at all pH levels (Fig. 1, curve 3); but when linoleic acid emulsion was added at pH 7.0, a rate of oxygen uptake occurred which was even higher than that produced when linoleic acid was added to the fresh homogenate. It could thus be concluded that the ether extraction and freezing treatments had no adverse effect upon the lipoxidase, but effectively removed the native substrates. When the results of this experiment are considered in conjunction with the demonstration of increased optical absorption at 234 rnp during oxygen absorption of the homogenate at pH 5.0 (Fig. 3, curves 1A and lB), and the results of the inhibition experiments (Table I), they strongly support the view that t’he oxygen absorption of the homogenate at pH 5.0 is due almost entirely t.o the lipoxidasesubstrate system. For the homogenate at pH 7.2, the inhibition of oxygen absorption by cyanide (Table I) indicated the participation of certain metallic oxidases in the oxygen uptake at this pH level. Cytochrome oxidase was detected in the homogenates from 214-day corn seedlings manometrically, using p-phenylenediamine as reductant (IO), and spectrophotometrically (2). However, since the homogenate itself was insensitive to azide (Table I), it is probable that cytochrome oxidase, even though present, does not contribute to the endogenous oxygen absorption of the homogenate. Phenoloxidase was also detected in homogenates from corn seedlings, but not at all ages. Using the thermolabile oxygen uptake induced by catechol as a measure of phenoloxidase, no activity was detected in homogenates from l-, 2-, and S-day corn seedlings. However, oxidation of catechol was rapidly catalyzed by homogenates from older seedlings (Fig. 4). Also, the production of intensely red compounds was observed during the course of oxygen absorption, a phenomenon associated with non-enzymatic reactions of quinoid products resulting from the oxidation of catechol (1, 3). This reddish pigment was faintly discernible with the use of homogenates from 2- or 3-day corn seedlings. Since phenoloxidase appears to be present only in small amounts in homogenates from such seedlings, and since oxygen uptake of homogenates was not strongly inhibited by diethyl dithiocarbamate (Table I), an inhibitor of this enzyme (8), it is unlikely that phenoloxidase is a major terminal oxidase in endogenous oxygen uptake of homogenates of 2x-day corn
444
GEORGE
FRITZ
AND
HARRY
BEEVERS
1507 .E 9 > 3
-
y IOO!T fj 0 508 P UQ
AGE OF SEEDLINGS (DAYS) Fm 4. Curve 1. The rate of oxygen absorption of homogenates from corn seedlings l-6 days old. The pH of the homogenates varied from 6.5 to 6.8, increasing with the age of the seedling. (One milliliter of the homogenate was used per flask, and KOH-saturated paper was in the center well.) Curve 2. The rate of oxygen absorption of the homogenates of curve 1 in the presence of 0.2 M catechol.
seedlings; however, the possibility is not excluded that phenoloxidase plays a greater role at later stages of development,. Still other metallic oxidases may contribute to the oxygen absorption of the homogenate at, pH 7.2. Although homogenates from corn seedlings can oxidize ascorbic acid, no conclusion was reached regarding the possible presence of ascorbic oxidase, since ascorbic acid can be oxidized through more than one enzyme system, such as cytochrome oxidase and phenoloxidase, as well as ascorbic oxidase (5, 9). The work of Ginter and Smith (4) indicated that a new type of oxidative enzyme may be present in homogenates from the tips of corn roots. It should be stressed that the conclusion that lipoxidase plays a major role in oxygen absorption at pH 5.0 applies only to the homogenates; it is not suggested that lipoxidase participates in the same degree in oxygen uptake of the intact tissue. Indeed, it was found that the respiration of 2x-day corn tissue was inhibited to the extent, of about, 50 % by application of 0.01 M cyanide. The clarification of the role of lipoxidase in the remaining (cyanide-insensitive) respiration must await further study.
LIPOXIDASE
The pH Optimum
AND
OXYGEN
-4BSORPTION
of the Lipoxidase-Catalyzed
Reaction
Holman and Bergstriim (7) have discussed the difficulties of determining the pH optimum of lipoxidase. Since the substrates for lipoxidase are difficultly soluble in water and not equally available to t,he enzyme at’ all pH \Talues, they concluded that the effect of pH upon (a) the inherent, enzyme activity and (b) the availability of t,he substrate to the enzyme cannot be separat,ed so long as emulsified substrates, not equally soluble over the entire pH range in question, are used. It, has already been shown that the lipoxidasa system a,rcounts for the bulk of the endogenous oxidative activity of homogenates from 21,i-day corn seedlings, and t)he pH optimum occurred at pH 5.0 (Fig. I, curve 1). However, when the homogenate was centrifuged (as previously described) and separated into two fractions, one of which contained lipoxidase and the other the substrates of lipoxidase, the pH optimum for the oxygen absorption process of the mixture of the two fractions occurred at pH 7.5, and very little oxygen was absorbed at pH 5.0. (When linoleic acid emulsion was added either to the supernatant or to the original (diluted) homogenate, the pH optimum was again 7.5.) Even if t)he sediment fraction was autoclaved, or subjected to wide cahanges in acidity before addition to the supernatant fract’ion containing lipoxidase, the pH optimum was still at pH 7.5 after mixture of the two fractions. Consequently, it was concluded that the centrifugation process itself was responsible for the loss of activity at pH 5.0, and the shift’ in optimum activity to pH 7.5. (In this connection, it may be noted that freezing and t,hawing of the corn seedlings had a similar effect, in t,hat t,hc optimum act,ivity had been shifted to a higher pH value; see Fig. 1, curve 2.) It is suggested that an intimate union between lipoxidase and its sub&rates exists in the homogenate prepared from fresh tissue, and accounts for the rapid endogenous oxygen absorption of the homogenate at pH 5.0. It would appear that this union is destroyed by physical treatments such as centrifugation, and is not completely restored upon subsequent, mixing of the enzyme and iDs substrat’es. (Probably the freezing and thawing process also partially disrupts t,his union.) Therefore it is suggested that the optimum pH value of the lipoxidase-catalyzed reaction in corn-seedling homogenates is pH 5.0. The fact that the pH optimum was found to be 7.5 when exogenous suhstrat,c was added, whether to t,he (diluted) homogenat,e or to the supernatant fraction, would be a result of t,he greater availability (nolubility) of the added substrate at t,he higher pH, but would not be a measure of the inherent
446
GEORGE
FRITZ
AND HhRRY
BEEVERS
pH optimum of the enzyme, a viewpoint already discussed by Holman and Bergstrijm (7). These comments indicate the need for distinguishing between native substrate already present and in closest possible contact with the enzyme, and exogenous or added substrate, limited in availability to the enzyme by its solubility. SUMMARY
Homogenates from 2x-day corn seedlings absorbed oxygen most rapidly at pH 5.0 and a second smaller maximum occurred at pH 7.2. The homogenat.es could be separated into t.wo fractions, one of which contained lipoxidase and the other contained substrates for lipoxidase. Several lines of evidence led to the conclusion that the lipoxidase-substrate system accounts for the major portion of the oxygen absorption of the homogenate at pH 5.0 but at pH 7.2, other enzymes systems must contribute to the oxygen absorption. REFERENCES 1. BEEVERS, H., ASD JAMES, W. O., Biochern. J. 43,636 (1948). 2. COOPERSTEIS, S. J., AND LAZAROW, A., J. Biol. Chem. 189, 665 (1951). 3. LAWSON, C. R., ASD TARPLEY, W. B., in “The Enzymes” (.I. B. Sumnerand K. Myrblick, eds.), Vol. II. .4cademic Press, New York, 1951. 4. GISTER, E. L., ASD SMITH, F. G., Zuwa Slate (‘011. J. Sci. 28, 177 (1953). 5. HII.L, R., AND HARTREE, E. F., Ann. Rev. Plant Physiol. 4, 115 (1953). 6. IIOLMAS, R. T., Arch. Biochem. 10, 519 (1946). 7. HOLMAN, R. T., ASD BERGSTRBM, S., in “The Enzymes” (J. B. Sumner and K. Myrb&ck, eds.), Vol. II. Academic Press, New York, 1951. 8. JAMES, W. O., Ann. Rev. Planl Physiol. 4, 59 (1953). 9. M~rsoN, I,. W., I’itamins and Hormones 11.1 (1953). 10. SLATEH, E. c., Biochem. .I. 44, 305 (1949). 11. THEORELL, H., RERGSTR~~M, S., .~SD AKESON, .4., Pharm. Acln Helv. 21, 318 (1946).